Sato O, Ogawa Y
Department of Pharmacology, Juntendo University School of Medicine, Hongo, Bunkyo-ku, Tokyo 113-8421, Japan.
J Biochem. 2001 Jun;129(6):881-9. doi: 10.1093/oxfordjournals.jbchem.a002933.
We suggested that an assembled form of phosphorylated myosin (P-myosin) might exhibit higher affinity for smooth muscle myosin phosphatase (SMMP) than dissociated P-myosin on the basis of the effect of MgATP [Sato and Ogawa (1999) J. Biochem. 126, 787-797]. To further deepen our understanding, we examined the SMMP activity and P-myosin assembly with various ionic strengths and Mg(2+) concentrations, with and without MgATP, all of which are well known to be critical for myosin assembly. The structure of myosin molecules was directly observed by electron microscopy using a rotary shadowing procedure, which was found to be consistent with the sedimentation assay. We found that the SMMP activity was always high when P-myosin was assembled. MgATP, which disassembled P-myosin mostly into a folded conformation, in contrast, decreased the enzyme activity. We also found that glycerol had a dissociating action on P-myosin, primarily dissociating it into an extended conformation, resulting in reduced SMMP activity, and that increases in the ionic strength and Mg(2+) (>5 mM) inhibited SMMP. These results indicate that myosin assembly is essential for SMMP activity.
基于MgATP的作用[佐藤和小川（1999年）《生物化学杂志》126卷，787 - 797页]，我们推测磷酸化肌球蛋白（P - 肌球蛋白）的组装形式可能比解离的P - 肌球蛋白对平滑肌肌球蛋白磷酸酶（SMMP）具有更高的亲和力。为了进一步加深我们的理解，我们在有和没有MgATP的情况下，研究了不同离子强度和Mg(2+)浓度下的SMMP活性和P - 肌球蛋白组装情况，所有这些因素都已知对肌球蛋白组装至关重要。使用旋转阴影法通过电子显微镜直接观察肌球蛋白分子的结构，发现其与沉降分析结果一致。我们发现当P - 肌球蛋白组装时，SMMP活性总是很高。相比之下，MgATP主要将P - 肌球蛋白分解为折叠构象，降低了酶活性。我们还发现甘油对P - 肌球蛋白有解离作用，主要将其解离为伸展构象，导致SMMP活性降低，并且离子强度和Mg(2+)（>5 mM）的增加会抑制SMMP。这些结果表明肌球蛋白组装对SMMP活性至关重要。
